Bilirubin is a naturally occurring bile pigment generated during the physiological breakdown of heme. Based on our findings that bilirubin ameliorates endotoxin-induced hepatotoxicity and suppresses allergen- induced pneumonitis, we postulate that bilirubin is an important physiological regulator of inflammation. The hypothesis to be tested by the proposed studies is that bilirubin is an endogenous inhibitor of inflammatory responses. The broad, long-term objectives of the outlined research are to characterize the cellular mechanism(s) underlying the anti-inflammatory properties of bilirubin and to explore potential therapeutic implications of these findings. The experiments outlined in Specific Aim 1 will investigate the role of bilirubin in the regulation of vascular adhesion molecule-1 (VCAM-1)- and intercellular adhesion molecule-1 (ICAM- 1 )-mediated leukocyte migration, with a specific focus on the inhibitory effect of bilirubin on endothelial NADPH oxidase and xanthine oxidase activity. Specific Aim 2 will focus on elucidating the mechanism(s) by which bilirubin prevents the up-regulation of inducible nitric oxide synthase (iNOS) in response to lipopolysaccharide (LPS) through an analysis of: 1) bilirubin-mediated suppression of c-Jun N-terminal kinase (JNK) activation;2) the modulatory effect of bilirubin on hypoxia inducible factor-1 (HIF-1) activity through suppression of NADPH oxidase-generated ROS and activation of the aryl hydrocarbon receptor (AhR);and 3) bilirubin-stimulated nuclear translocation of the constitutive androstane receptor (CAR). The studies proposed under Specific Aim 3 are designed to examine the ability of leukocytes and endothelial cells to generate and metabolize bilirubin in situ. In summary, the experiments proposed in the present application are designed to systematically characterize the mechanism(s) by which bilirubin suppresses inflammatory responses. It is anticipated that the proposed experiments will provide new insights into the role that bilirubin plays in the regulation of inflammation. As bilirubin is generally innocuous and, as the levels of serum bilirubin associated with anti-inflammatory effects are modest (20 uM =1.2 mg/dL), we are hopeful that the results of these investigations will lay the foundation for potential new and effective therapies for the treatment of a variety of inflammatory conditions.